Optimized Incorporation of Alkynyl Fatty Acid Analogs for the Detection of Fatty Acylated Proteins using Click Chemistry

This method improves the delivery of fatty acids to cells and protects them from the toxic effects of free fatty acid delivery, thus ensuring more consistent labeling. The sensitivity and efficiency of click chemistry detection are dependent on the effective uptake of the fatty acid label. We have shown that this can be largely improved.

Several steps of the protocol are very specific and need to be followed closely. A visual demonstration can clarify and show, for example, how to avoid formation of solids in the labeling mix. To prepare the labeling media, supplement DMEM with 5%dextran/charcoal coated FBS, 1X penicillin streptomycin, two millimolar L-glutamine, and 100 millimolar sodium pyruvate, then prewarm it to 37 degrees Celsius before use.

Gently wash the HEK-293 T-cells plated one day prior in a six-well tissue culture dish with PBS, then replace the PBS with labeling media. Incubate the cells at 37 degrees Celsius with 5%carbon dioxide for approximately 45 minutes before proceeding to metabolic labeling with fatty acids. To saponify the fatty acid analogs, pipette at least two microliters of the alkynyl fatty acid analog directly into the bottom of a three milliliter conical reaction vial.

Pipette an equal amount of diluted potassium hydroxide close to the bottom of the reaction vial on the edge of the glass such that the dispensed volume of the potassium hydroxide mixes with the fatty acid. Close the lid of the vial and tap gently to mix the solutions. Heat the reaction vial at 65 degrees Celsius for approximately five minutes or until the solution becomes clear, indicating that the fatty acid has been incorporated.

However, take care that the liquid does not evaporate too much. Next, pipette pre-warmed 20%fatty acid-free BSA such that the volume ratio of fatty acids to potassium hydroxide to fatty acid-free BSA is 1-1-50, achieving a final concentration of 20X BSA-bound alkynyl fatty acids. Mix the solution by pipetting up and down, then incubate it for 15 minutes at 37 degrees Celsius.

Label the HEK-293 T-cells by adding the 20X fatty acid BSA conjugate directly into the starvation media to achieve a final concentration of 1%BSA in 25 micromolar alkynyl myristate or 100 micromolar alkynyl palmitate and alkynyl stearate. For comparison, add non-saponified liquids by pipetting two microliters of unlabeled fatty acid directly into the starvation media, then place the cells back into the incubator for three to six hours. After the incubation is complete, gently wash the cells with PBS at room temperature, then harvest and lyse the cells by adding 500 microliters EDTA-free modified RIPA buffer and rotating the lysates for 15 minutes at four degrees Celsius.

Centrifuge the lysates at 16, 000 times G for 10 minutes at four degrees Celsius, then collect the supernatant in 1.7 milliliter microcentrifuge tubes and store them at minus 20 degrees Celsius. Bring 50 to 100 micrograms of protein lysates in 1.7 milliliter microcentrifuge tubes to an equal volume using the EDTA-free modified RIPA buffer. Keep the reaction volume as small as possible.

Add STS to each sample to attain a final concentration of 1%Prepare a master mix of the click reagents and add appropriate volumes into the lysates, then mix by pipetting up and down. Incubate the lysates in the dark for 30 minutes in a 37 degree Celsius water bath with occasional mixing. For immunoprecipitation, mix lysates with rabbit anti-GFP and incubate overnight with rotation at four degrees Celsius.

Add 15 to 20 microliters of magnetic beads pre-equilibrated with 0.1%SDS RIPA to each tube and allow it to react end over end at four degrees Celsius for three hours. Wash the beads with lysis buffer and resuspend in 45 microliters of 50 millimolar HEPES buffer containing 1%SDS. Heat the beads at 80 degrees Celsius for 15 minutes.

During the incubation, invert or agitate the tubes approximately every five minutes, then briefly spin all the tubes. While the samples are still warm, collect the supernatant containing the proteins. Combine 43 microliters of the supernatant with seven microliters of the click reagents master mix and allow them to react in the dark for 30 minutes at 37 degrees Celsius.

On a Western plot, a noticeable effect was observed in labeling efficiency for click chemistry detection between saponified and non-saponified alkynyl fatty acids with an increasing length of the acyl chains. In cells labeled with alkynyl stearate, saponification of the fatty acid and delivery with BSA for metabolic labeling drastically increased the detection of S-acylated protein signal through click chemistry and detection by a fluorescent azitoprobe, suggesting an overall increase in cellular incorporation of the alkynyl fatty acid label. Conversely, no noticeable difference was observed in cells treated with the shortest and most soluble fatty acid alkynyl myristate.

Cells labeled with alkynyl palmitate showed an intermediate increase in label compared to alkynyl myristate, but less than alkynyl stearate. Importantly, treatment of PVDF membranes with 0.1 molar potassium hydroxide largely removed the fatty acid labels from cells incubated with alkynyl palmitate and the alkynyl stearate, confirming that the majority of the signal was through an ester or thioester bond. As expected, the incorporation of alkynyl myristate was mostly alkali resistant due to the attachment of myristate to proteins through an amide bond.

Following click chemistry, myristoylation of wild type myristoylated C-terminal Huntington GFP was detected in the immunoprecipitates, as well as the lysates, whereas the G2A mutation completely blocked myristoylation of the C-terminal Huntington GFP. Following click chemistry, we can perform affinity purification of fatty acylated proteins for mass spectrometry. This may yield a different profile of fatty acylated proteins by protecting cells from toxicity.